How information is encoded, processed, and transmitted by the nervous system is far from completely understood. Most neurons use a single neurotransmitter to transmit information to downstream neurons. However, it is now widely accepted that there is a subset of neurons that utilize two distinct neurotransmitters. Most studies on dual neurotransmitter usage have, however, focused on establishing that the phenomenon is real, with comparatively little attention given to the underlying functional purpose. Recent experiments in this laboratory have identified a subset of ~100 interneurons (~1% of total) in the Drosophila larval nervous system that use both acetylcholine and glutamate. To understand the biological role of these neurons, directly connecting upstream and downstream neurons will be identified using an improved synapse-specific GFP Reconstitution Across Synaptic Partners (GRASP) method recently developed in this laboratory to screen a larval-expressing subset (419) of a 7000 strain collection of GAL4 drivers developed for fine-scale neural circuit mapping. Once the upstream and downstream neurons have been identified, what types of neurons they are will be discerned based on neuroanatomy since sensory, motor, and interneurons have distinct morphologies. Knowledge of the types of neurons upstream and downstream will place the acetylcholine/glutamate dual neurotransmitter neurons in a biological context and thereby provide insights into the direction of information flow, the type of informatio they are carrying, and the demands of information transfer. To understand the functional purpose of the acetylcholine/glutamate dual neurotransmitter neurons, cholinergic and glutamatergic transmission will be eliminated, singly and in combination, specifically in these neurons using existing mutations and the consequences will be assayed behaviorally and by functional calcium imaging. Larval behavioral responses to light, mechanosensation, and temperature will be assayed. Since these different stimuli each activate distinct sensory and motor circuits, these behavioral assays will together assess a substantial portion of the larval nervous system. The single or dual silenced Ach/vGlut dual neurotransmitter neurons will be excited optogenetically and the activation of their downstream neurons will be assessed using genetically-encoded calcium indicators. The results of these experiments will test the hypothesis that distinct types of information, such as stimulus intensity and stimulus direction, or distinct modes of transmission, such as fast excitatory and modulatory, are being carried by acetylcholine and glutamate within the same neurons.